Channel kinases TRPM6 and TRPM7 belong to the alpha-kinase family, unusual protein serine-threonine kinases that do not display sequence homology to conventional eukaryotic protein kinases. Channel kinases are unusual bifunctional molecules consisting of a protein kinase domain fused to an ion channel. These molecules play a key role in the regulation of magnesium homeostasis in vertebrates. The TRPM7 channel has been characterized using electrophysiological techniques, however the role of the kinasedomain remains unknown. We generated knockout mouse with a disruption of the TRPM7 kinase domain and found that this results in early embryonic lethality, suggesting that the kinase domain has an essential and non- redundant function. We have recently identified annexin 1 as an endogenous substrate for TRPM7.We found that TRPM7 kinase phosphorylates annexin 1 at a conserved serine residue located within an amphipathic alpha-helix that plays a crucial role in annexin function. Our preliminary experiments suggest that TRPM7 kinase can phosphorylate myosin IIA and several other substrates. Our experiments also suggest that TRPM7 and TRPM6 kinases utilize an unusual mechanism of substrate recognition. Our goal is to determine physiological function of TRPM7 and TRPM6 kinase domains, to elucidate the mechanism of their activation and substrate recognition and the role in the regulation of channel activity. Therefore the specific aims are: 1) To identify physiological substrates and to determine sequence motif recognized by TRPM7 and TRPM6 kinases. To investigate mechanism of autophosphorylation of TRPM7 and TRPM6 kinases, to map autophosphorylation sites, and to investigate the role of autophosphorylation in the regulation of channel activity (in collaboration with Dr. Fleig, Project 1). The role of identified kinase substrates in magnesium- dependent signaling will be investigated (in collaboration with Dr. Scharenberg, Project 3). 2) To investigate mechanism of substrate recognition and to test the hypothesis that TRPM7 andTRPM6 kinases recognize alpha helical conformation in its substrates. 3) To determine the kinetic mechanismof TRPM7 and TRPM6 kinases, to elucidate the mechanism of their activation, and to investigate the role of divalent metal ions in the modulation of their activity.